Multiple studies suggest that measurement of indicators of platelet activation might offer advantages in the clinical evaluation of patients at risk from thrombotic and other diseases (Macey, M. G., Carty, E., Webb, L., Chapman, E. S., Zelmanovic D., Okrongly, D., Rampton, D. S., Newland, A. C. (1999) “Use of mean platelet component to measure platelet activation on the ADVIA 120 Haematology System” Cytometry, 38, 250–255.; Mody, M., Lazarus, A. H., Semple, J. W. Freedman, J. (1999) “Pre-analytical requirements for flow cytometric evaluation of platelet activation: choice of anticoagulant.” Transfusion Medicine 9, 147–154).
Mean platelet component (“MPC”) is a parameter which can be determined by standard laboratory haematology analysers, such as the commonly used ADVIA 120™ Haematology System, produced by Bayer AG. MPC is a measure of mean refractive index of the platelets, and its value as a measure of platelet activation has been previously suggested in the Macey et al. paper referred to above. In vitro stimulation of normal platelets in whole blood by bovine thrombin resulted in activation leading to increased platelet expression of CD62P (a measure of activation) and a concomitant decrease in MPC. This response was dose and time dependent.
The ADVIA 120 Haematology System has a laser optical bench that consists of a laser diode, a flow cell and detector assemblies. A laser diode is used to produce monochromatic light focused onto the flow cell. According to the Mie Scattering Theory of light, the intensity of monochromatic light scattered at a particular angle by a uniform homogeneous sphere depends only on its volume and the average refractive index (RI) difference between the sphere and the medium in which it is suspended. This provides an equation for the intensity of monochromatic scattered light within given angular intervals that is a function of only two unknowns, volume and RI. Measuring the light scattering at two appropriate different angular intervals provides two equations with two unknowns that can be solved numerically. This is the basis of the ADVIA 120 Haematology System red blood cell and platelet measurements (Tycko, D. H., Metz, M. H., Epstein, E. A., Grinbaum, A. (1985) “Flow cytometric light scattering measurement of red cell volume and hemoglobin concentration.” Applied Optics, 24, 1355–1360; Zelmanovic, D., Colella, G. M., Hetherington, E. J., Chapman, S. E., Paseltiner, L. (1998) “Automated method and device for identifying and quantifying platelets and for determining platelet activation state using whole blood samples.” U.S. Pat. No. 817,519; Kunicka, J. E., Fischer, G., Murphy, J. and Zelmanovic, D. (2000) “Improved platelet counting using two-dimensional laser light scatter.” American Journal of Clinical Pathology 114, 283–289).
The ADVIA 120 determines both the volume and RI of platelets on a cell-by-cell basis. Platelets intercept an incident beam of monochromatic 675±10 nm laser light emitted by a photodiode as they pass through an optical flow cell. The system measures the intensity of light scattered in the ranges of 2–3 degrees and 5–15 degrees. The pair of scattering-light intensity values is transformed into particle volume and RI values by reference to a look-up table based on the Mie Scattering Theory for homogeneous spherical particles. The platelet RI value is converted to the Mean Platelet Component (MPC) concentration, by subtracting the index of refraction of water (1.333) from RI and dividing the difference by the average refractive index increment (0.0018 dL/g) (Zelmanovic et al., 1998). This constant is derived from the weighted average refractive index increments for the major components of platelets, namely protein, lipid, and carbohydrate (Armstrong, S. H., Budka, M. J. E., Morrison, K. C., Hasson, M. (1947) “Preparation and properties of serum and plasma proteins. The refractive properties of the proteins of human plasma and certain purified fractions.” Journal of the American Chemistry Society, 69, 1747–1753; Barer, R., Joseph, S. (1954) “Refractometry of living cells.” Quarterly Journal of Microscopical Science, 95, 399–423; Zelmanovic et al., 1998). Recent evidence also indicates a linear relationship between platelet density (separated by stractan gradients) (Corash, L., Tan, H., Gralnick, H. (1997) “Heterogeneity of human whole blood platelet sub-populations. I. Relationship between buoyant density, cell volume, and ultrastructure.” Blood, 49, 71–87) and MPC (Chapman, E. S., Lerea, K. M., Kirk, R., Sorette, M. P., Sanjay, N. S., Zelmanovic, D. (1998) “Monitoring in vitro and ex vivo platelet activity: comparison of alpha granule release, density distribution, platelet adhesion and mean platelet component concentration (MPC).” Blood, 92, Suppl. 1, 68B, Abstract 3273).
Mean platelet mass (MPM, pg) is computed from the mean PV (MPV) and MPC. Date for individual platelets may be presented in cytograms while the mean value for each parameter is tabulated (Zelmanovic, D., Colella, G. M., Hetherington, E. J., Chapman, E. S., Paseltiner, L. (1998) “Automated method and device for identifying and quantifying platelets and for determining platelet activation state using whole blood samples”. U.S. Pat. No. 5,817,519).
Platelet activation occurs swiftly upon venesection or promptly afterwards, and this characteristic of platelets makes ex vivo analyses difficult. However, the extent of this in vitro activation does depend on the anticoagulant into which the blood is collected (Kuhne, T., Hornstein, A., Semple, J., Chang, W., Blanchette, V., Freedman, J. (1995) “Flow cytometric evaluation of platelet activation in blood collected into EDTA vs. Diatube-H, a sodium citrate solution supplemented with theophylline, adenosine, and dipyridamole.” American Journal of Hematology,50, 40–45). For reasons of simplicity and economy, the ideal anticoagulant would be one that enabled information on platelet activation to be obtained as part of the full blood profile.
Ethylenediaminetetra-acetic acid (EDTA) as its liquid tripotassium salt is commonly used as an anticoagulant due to its availability and convenience to prepare (Perrotta, G., Roberts, L., Glazier, J., Schumacher, H. R. (1998) “Use of sodium citrate anticoagulant for routine hematology analysis on the CELL-DYN® 4000: An opportunity to enhance efficiency in the clinical laboratory.” Laboratory Hematology, 4, 156–162). It is also the preferred anticoagulant for complete blood counts and white blood cell differentials because of its cell preservation properties, and is the anticoagulant recommended for these purposes by the National Committee for Clinical Laboratory Standards (NCCLS; standard H1-A4).
When monitoring platelet activation ex vivo, the main requirements are to use a venepuncture procedure that minimises spontaneous platelet activation and to collect blood into a medium that not only prevents coagulation, but will also preserve the activation status of platelets until the sample can be analysed (George J N, Thio L L, Morgan R K (1981) “Quantitative analysis of platelet membrane glycoproteins; effect of platelet washing and isolation of platelet density subpopulations.” Thrombosis Research 23, 69–77; Hawiger J (1989) “Platelet secretory pathways: an overview.” Methods in Enzymology 169, 191–195; Michelson A D (1996) “Flow cytometry: a clinical test of platelet function—a review.” Blood 87, 4925–4936; Wu K K (1994) “Platelet activation and arterial thrombosis.” Lancet 344, 991–995).
For platelet investigations EDTA is unsuitable as it causes them to swell, and experience auto-activation, which increases over time (Kuhne et al., 1995; Jackson, S. R., & Carter, J. M. (1993) “Platelet volume: Laboratory measurement and clinical application.” Blood Reviews, 7, 104–113; McShine, R. L., Das, F. P. C., Siblinga, C. S., Brozovic, B. (1990) “Differences between the effects of EDTA and citrate anti-coagulants of platelet count and mean platelet volume.” Clinical and Laboratory Haematology, 12, 277–285; Pidard, D., Didry, D., Kunicki, T. J., Nurden, A. T. (1986) “Temperature-dependent effects of EDTA on the membrane glycoprotein IIb/IIIa complex and platelet aggregability.” Blood, 67, 604–611). The gradual swelling of platelets is due to alterations in the plasma membrane induced by EDTA and results in a fall in optical density and an increase in mean platelet volume (MPV). The fall in optical-density may be measured on the ADVIA 120 system (Bayer AG) as a change in the mean platelet component (MPC) (Zelmanovic et al., 1998). EDTA also alters the morphology of platelets from their innate ellipsoidal shape to spherical. Platelet shape change is a rapid reaction that can be measured either by morphologic methods such as scanning electron microscopy or by following the increase in optical density that occurs in the aggregometer. The optical technique indicates that ADP-induced shape change reaches the half-maximal optical density change in 2.5 seconds. Platelet shape change is characterised by a morphologic transformation from the normal discoid (2 to 4 μm in diameter and about 0.5 μm thick) shape to a spiny sphere containing many long, thin filopodia. This shape change begins immediately upon exposure to the anticoagulant and is maximal within 2 hr (Jackson and Carter, 1993). These changes are detectable by the ADVIA 120 after 30 minutes. Furthermore, if blood from certain individuals is anticoagulated with EDTA, the platelets aggregate, causing an apparent thrombocytopenia to be recorded (Okada T (1999) “development and problems of automatic haematology analysers.” Sysmex Journal International 9, 52–57).
Therefore for platelet studies trisodium citrate has been established as the anticoagulant of choice for platelet studies (Perrotta et al., 1998). It has been demonstrated that citrate-comprising anticoagulants give rise to a lower MPV than EDTA, a trait partly explained by the preservation of normal discoid morphology (Jackson and Carter, 1993). When blood is collected in citrate, there is initially little or no change in platelet shape and volume. However, in citrate, the platelets slowly adopt a spherical shape (Macey et al., 1999) and, as in EDTA, swell progressively over a period of 1–2 h (3–10% increase in volume by impedance procedures depending on the concentration of the sodium citrate (Bath, PWM (1993) “The routine measurement of platelet size using sodium citrate alone as the anticoagulant.” Thrombosis and Haemostasis 70, 687–690; Threatte G A, Adrados C, Ebbe S, Breecher G. (1984) “Mean platelet volume. The need for a reference method.” Am J Clin. Pathol. 81: 769–72). For these reasons citrate was originally considered unreliable for the measurement of platelet volume (Thompson C B, Diaz D D, Quinn P G, Lapins M, Kurtz S R, Valeri C R. (1983). “The role of anticoagulation in the measurement of platelet volumes.” Am J Clin Pathol 180: 327–32).
Citrate-based anticoagulants have been used for the determination of platelet parameters in the ADVIA 120 (Macey et al., 1999; Zelmanovic et al., 1998). However, the standard deviation of the MPC (recorded as platelet component distribution width (PCDW)) is initially greater in citrate than EDTA, because the light scatter characteristics of disc-shaped platelets, unlike that of spheres, is dependent on their orientation (Macey et al., 1999; Zelmanovic et al., 1998).
In previous studies it has been found that when blood anticoagulated with either sodium citrate (Maurer-Spurej, E., Pfiefer, G. Maurer, N., Linder, H., Glatter, O., Devine, D. V. (2001), “Room temperature activates human blood platelets”. Lab. Invest. 81, 581–592) or acid citrate dextrose (ACD) (Oliver, A. E., Tablin, F., Walker, N. J., Crowe, J. H. (1999) “The internal calcium concentration of human platelets increases during chilling”. Biochimica Biophysica Acta, 1416, 349–360) was cooled to 20° C. and 5° C. respectively the platelets were found to be activated spherical cells with pseudopodia. We have shown (unpublished data) in blood anticoagulated with sodium citrate incubated at 4° C. that there is a time dependent increase in CD62P expression on platelets and a significant increase in PLA formation.
A pilot study using the Abbott CEL L-DYN 4000™ haematology system indicates that citrate can be used instead of EDTA for routine full blood cell counts, provided that corrections are made to take account of the different dilution factor.
O'Malley et al. (“Measurement of platelet volume using a variety of different anticoagulant and antiplatelet mixtures”, Blood Coagulation and Fibrinolysis 7 (4), 1996, 431–436) disclose an anticoagulant containing EDTA and theophylline for use in measuring mean platelet volume.
A relatively new anticoagulant commercially named Diatube-H™ has been developed to inhibit platelet activation and has been used for measuring plasma heparin levels and platelet factor 4 and β-thromboglobulin release from activated platelets (Kuhne et al., 1995). The main constituents of Diatube-H™ are citrate, theophylline, adenosine and dipyridamole, and it is informally termed CTAD. Theophylline and dipyridamole have been shown to inhibit phosphodiesterase activity, which leads to an increase of platelet cyclic AMP and a reduction in platelet activity. Adenosine also inhibits thrombin-induced platelet aggregation and release of intracellular calcium (Kuhne et al., 1995). Dipyridamole has the disadvantage of being light sensitive and CTAD anticoagulant tubes should be stored appropriately.
Platelets in EDTA approximate homogeneous spheres for the purposes of applying Mie theory and obtaining accurate volume and MPC values on a cell-by-cell basis (Zelmanovic et al., 1998). However, because of the platelet activating property of EDTA it is generally thought to be highly unsuitable for use as an anticoagulant for measuring patient platelet activation status.
Measurement of neutrophil activation is also of clinical importance. Neutrophils show little or no evidence of activation (e.g. by their level of CD11b expression) if analysed in whole anticoagulated blood shortly after venesection. Unfortunately, both EDTA and citrate decrease the Ca2+ concentration in plasma and consequently affect the antigenicity of Ca2+ dependent epitopes such as CD11b. An alternative reagent, Cyto-Chex™ (Streck Laboratories, Omaha, Nebr., USA), that is recommended for the preservation of whole blood, stabilises antigen expression on lymphocytes and neutrophils but little is yet known of its effect on platelets.